Effects of tumour acidification with glucose+MIBG on the spontaneous metastatic potential of two murine cell lines

Abstract

In addition to hypoxia, acidic extracellular pH (pH(e)) is recognised as one of the microenvironmental characteristics of solid tumours. A number of studies have examined ways to increase tumour acidity in order to improve tumour-specific targeting of certain drugs and the effectiveness of hyperthermia. However, previous data have shown that exposure of murine tumour cells to acid conditions in culture can enhance their metastatic potential when injected subsequently into mice, raising the concern that deliberate tumour acidification might increase the probability of metastasis. In this study, we examined the effects of in vivo tumour acidification and hypoxia on the spontaneous metastatic potential of the murine KHT-C fibrosarcoma and B16F1 melanoma cell lines. A tumour-specific increase in extracellular acidity, demonstrated by measurements with pH electrodes, was achieved by daily intraperitoneal injections of meta-iodo-benzylguanidine (MIBG) and/or glucose. This method of tumour acidification during tumour growth did not significantly enhance the spontaneous metastatic potential of the two murine cell lines.

Figure 1

Histograms of pooled pH_e values before and after treatment in KHT-C and B16F1 tumours. (A) KHT-C: Control: pretreatment measurements from animals used for post-treatment measurements, obtained 1 day prior to or on the day of treatment. Mean pH_e=7.13±0.017; N=25; n=179. Glucose+MIBG: Animals treated with 3 g kg⁻¹ glucose+30 mg kg⁻¹ MIBG were measured 1 h postinjections. Mean pH_e=6.89±0.030; N=12; n=99. Acute hypoxia: mice were measured during reoxygenation cycles of 160 min of acute hypoxia treatment. Mean pH_e=7.23+0.045, N=8; n=51. Acute hypoxia+glucose+MIBG: animals were injected with one dose of glucose+MIBG prior to acute hypoxia and were measured as described for acute hypoxia alone. Mean pH_e=6.97±0.031; N=5; n=32. (B) B16 F1: animals were measured as described for the KHT-C tumours, unless specified otherwise. Control: all pretreatment measurements were made on the day of the first treatment. Mean pH_e=7.14±0.022; N=19; n=152. Glucose+MIBG: measurements were carried out following 1–3 rounds of injections on days 7–9. Mean pH_e=6.89±0.064; N=6; n=48. Acute hypoxia: mean pH_e=7.01±0.063; N=4; n=33. Acute hypoxia+glucose+MIBG: mean pH_e=6.79±0.042; N=4; n=36. (mean±1 s.e.m.; N=the number of animals used; n=pooled number of pH readings obtained).

Figure 2

Kinetics of pH_e reduction following tumour-acidification treatments on four consecutive days in KHT-C and B16 F1 tumours. The distribution of pH_e was measured daily in total of seven different tumour-bearing animals. One animal on day 8 in panel A was omitted due to a missed time point and another animal on day 10 in panel B died. The three connected circles for each animal represent pre-, 1 h post- and 3 h postinjection time points. Maximal reduction in mean pH_e of both tumour models was generally observed at the first time point, 1 h post-treatment and pH_e returned near the initial value by 3 h post-treatment. (A) KHT-C (N=7): pretreatment measurements were made on each day of the treatment during days 7–10. Injections consisted of 3 g kg⁻¹ glucose+30 mg kg⁻¹ MIBG on days 7 and 9 and of 3 g kg⁻¹ glucose on days 8 and 10. (B) B16F1 (N=7): all pretreatment measurements were obtained on day 7. The animals were treated with 3 g kg⁻¹ glucose+30 mg kg⁻¹ MIBG on days 7–9 and with 3 g kg⁻¹ glucose alone on day 10.

Figure 3

Oxygen tension measurements in KHT-C and B16F1 tumours treated with 30 mg kg⁻¹ MIBG+3 g kg⁻¹ glucose. Most tumours (seven out of eight) were concurrently monitored with two OxyLite probes. No consistent changes in pO₂ levels in response to the injections were observed. (A) KHT-C tumours (N=4); measurements were started within 30 min of injections and continued up to 3 h after treatment. (B) KHT-C tumours (N=2); measurements were started prior to injections, marked by • and , and continued for 1–2 h after treatment. (C) B16F1 tumours (N=2); measurements were started prior to injections, marked by • and , and continued up to 2 h after treatment.

Figure 4

Microscopic lung metastases detected in KHT-C or B16F1 tumour-bearing animals treated with glucose+MIBG (□), glucose (⋄) or control injections (PBS, ▵) in combination with acute hypoxia or control air gassing conditions, indicated by solid and open symbols, respectively. (A) KHT-C: pooled medians from 2–4 repeat experiments (R): Air, PBS=17 (4R, N=33); Air, glucose+MIBG=20 (4R, N=33); Air, glucose=20 (2R, N=23); Hypoxia, PBS=18 (4R, N=28); Hypoxia, glucose+MIBG=24 (4R, N=29); Hypoxia, glucose=25 (2R, N=17). (B) B16F1: medians from one set of experiments: Air, PBS=1 (N=15); Air, glucose+MIBG=2 (N=15), Hypoxia, PBS=2 (N=14); Hypoxia, glucose+MIBG=1 (N=13).